We propose to synthesize full-length transcripts of DNA complementary (cDNA) to genomic RNA of avian and murine RNA tumor viruses. Full-length cDNA will be used as a template to direct the synthesis of the second strand of proviral DNA. The mechanism of synthesis of the second strand of DNA will be investigated. The in vitro synthesized single-stranded and double-stranded DNA will be cleaved by restriction endonucleases to generate specific fragments. The order of the fragments will be determined with respect to the 3'-end and 5'-end of the genome. Restriction enzyme digested in vitro synthesized proviral DNA from various viruses will be compared to construct a physical map of the viral genome. The regions of nucleic acid sequence homology and nonhomology among various strains of murine leukemia viruses and murine sarcoma viruses will be determined by heteroduplex formation. The heteroduplexes will be analyzed by visualization under an electron microscope. In vitro synthesized DNA will be assayed for infectivity. DNA transcripts representing part of the genome will be assayed for infectivity and viral gene expression. The map position of the temperature-sensitive mutants will be determined by marker rescue with restriction enzyme fragments obtained from DNA synthesized with wild-type virions. Attempts will be made to construct in vitro mutants with deletions at specific positions. Both the single-stranded and double-stranded DNA synthesized in vitro will be transcribed with DNA-dependent RNA polymerases. DNA fragments generated by restriction endonucleases will be used to direct protein synthesis in a linked transcription-translation system.